Myosins are a superfamily of actin-based molecular motors, ubiquitous in animal cells. The cyclic, ATP-hydrolysis-driven interaction of myosin with filamentous actin (F-actin) has been implicated in a variety of intracellular functions, including cell migration and adhesion;intracellular transport and localization of organelles and macromolecules;signal transduction;and tumor suppression. The importance of these interactions is illustrated by the identification of disease-causing myosin mutations, manifested in development defects as well as cardiovascular and neuronal diseases. The basic mechanism by which all myosins interact with actin is generally conserved, but different myosins have tuned their structural, kinetic, and mechanical properties to optimize performance for their particular cellular role. We hypothesize that the modes in which myosins adapted, correspond to specific "structural signatures", which we aim in determining here using electron cryo-microscopy and advanced computational imaging techniques, focusing on class II myosins, in the context of the cardiac system. Since the inception of the grant, our studies showed that myosin loops at the actin interface (loop 2), at the nucleotide-binding pocket (loop 1), and a large cleft that divides the actin-binding region of myosin are key structural elements that determine the mode in which myosin binds actin during the hydrolysis cycle. Our studies provided detailed residue-based actomyosin interface information for the strong-binding states. We showed the existence of a structural correlate to the postulated strain-dependent ADP release mechanism in smooth muscle myosin that does not exist for skeletal myosin. Such a release mechanism would benefit a myosin designed for high forces and slow contractions. Finally, we provided a detailed structural mechanism for myosin V processivity which included the determination of two structures of previously inaccessible weakly-bound actomyosin states one of which shows the lever-arm in an 'up'position for the first time. In this proposal, we will continue developing and using state-of-the-art EM image reconstruction approaches and use an array of specifically selected myosins and actins to provide (a) high-resolution structure of F-actin, (b) a detailed, residue-level actomyosin interface information (c) provide "structural signatures" directly associated with the two cardiac myosin isoforms, alpha- and beta-cardiac, in the context of the actomyosin assembly and disease-causing mutations associated with familial hypertrophic cardiomyopathy. PUBLIC HEALTH REVELANCE: Heart failure is a world wide public health problem that affects several million patients in the United States alone. One prime cause of heart disease is familial hypertrophic cardiomyopathy (FHC), which is an inherited cardiac disease that frequently results in sudden death of young and otherwise healthy individuals. Here, we propose using a combination of advanced imaging and computational techniques to provide an in-depth characterization of the molecular motors intimately involved in normal and aberrant (FHC) heart function. Our studies will provide new perspectives to consider for targeted therapeutic diagnostics or intervention.